Abstract Body

The HIV-1 mature conical core, composed of ?250 capsid protein (CA) hexamers and ?12 pentamers, must disassemble (uncoat) and the viral DNA must enter the nucleus before it can integrate into the host genome. For the past 4 decades, retroviral uncoating has been widely believed to occur in the cytoplasm, and some recent studies have proposed that uncoating occurs at the nuclear envelope (NE) just prior to nuclear import. Studies of uncoating have been hampered by an inability to accurately quantify the amount of CA associated with viral reverse transcription/preintegration complexes, and an inability to study rare infectious viral cores in a vast majority of non-infectious viral cores. We recently developed methods to directly label CA with green fluorescent protein (GFP) and track viral cores in infected cells by live-cell microscopy. In addition, we developed methods to identify infectious viral cores that led to the formation of transcriptionally active proviruses. In striking contrast to the prevailing models of nuclear import and uncoating, our results showed that infectious viral cores in the nucleus are intact and complete reverse transcription in the nucleus before uncoating. Recent studies from other groups supporting this model have shown that viral cores in the nucleus are largely intact, that reverse transcription requires an intact capsid and that reverse transcription is completed in the nucleus. We also probed the mechanism of viral core nuclear import and showed that intact viral cores gain nuclear entry through a mechanism involving interactions at the NE with the host protein cleavage and polyadenylation specificity factor 6 (CPSF6). Using GFP as a capsid content marker, we have recently observed that nuclear capsids retain their integrity and maintain a separation between the viral core contents and the nuclear environment until <1.5 hours before integration. These observations fundamentally change our current understanding of HIV-1 post-entry replication events including mechanisms of nuclear import and uncoating as well as reverse transcription, integration, and evasion of innate immunity.